Extracorporeal photopheresis is a process where 8-Methoxypsoralen (8-MOP), a naturally occurring light-sensitive compound, is administered orally two hours prior to treatment; blood is then withdrawn from the patient, anticoagulated, and the white blood cells are separated by centrifugation and collected as a leukocyte enriched fraction. These 8-MOP containing leukocytes are then irradiated with ultraviolet A light (UVA) which binds the 8-MOP to pyrimidine bases in DNA and to intra- and extra-cellular proteins. These treated leukocytes are returned to the patient, and the result is an immunomodulation which has been found to be of clinical benefit in a number of disease states.sup.1.
There are a number of diseases which are felt to primarily involve T-cells or are T-cell mediated. Diseases such as cutaneous T-cell lymphoma, organ allograft rejection after transplantation, progressive systemic sclerosis (PSS), inflammatory bowel disease (IBD), rheumatoid arthritis (RA) and juvenile onset diabetes mellitus (JODM) are thought to be T-cell mediated.
Cutaneous T-cell lymphoma (CTCL) is a malignant disease that is progressive. Therapeutic options are limited. Edelson et al. performed a multi-center trial.sup.2 which showed that 24 of 29 (83%) of erythrodermic patients experienced a significant improvement in their disease. These positive responses were seen at a median time of 22.4 weeks after initiation of therapy. Of clinical significance, these patients were those whose diseases were resistant to prior therapy which is felt to be a poor prognostic group. In addition, a decrease in the amount of peripheral blood involvement (Sezary cells) was seen. Actuarial data had indicated that median survival was increased to greater than 60 months from the onset of treatment in comparison with a historical median survival time of less than 30 months. In this original group of patients, remissions were sustained in eight of the subjects who were leukemic. Adverse reactions associated with photopheresis were rare.
Autoimmune diseases are characterized by a dysregulation of the immune system, characterized by specific cellular or humoral mediated destruction of specific organs or tissues in the patient. Examples of such diseases are rheumatoid arthritis and progressive systemic sclerosis.
Rheumatoid arthritis (R.A.) is an inflammatory disease that ultimately leads to joint destruction and is a generalized disease involving many organ systems. There are many pharmaceutical agents in use for R.A., however well tolerated agents with disease modifying potential are needed in as much as the disease is lifelong. In particular, a loss of efficacy and disease progression is seen in a high number of patients after starting secondary line therapy for R.A. Many of the second line agents are immunosuppressive and are themselves the reason for the major side effects such as infection. The need for development of a more specific, non-toxic immunomodulating therapy.sup.3 is great.
Progressive systemic sclerosis (PSS) is a connective tissue disease characterized by inflammatory and fibrotic changes in the skin and viscera. Treatment has been difficult. Anti-inflammatory drugs and corticosteroids are helpful in the early stages of the disease, but do not appear to influence the progression of the disease. Trials with D-penicillamine, methotrexate, cyclosporine, calcium channel blockers and prostagladins are underway, but these agents do not appear to influence the overall progression of the disease. As this disease has been considered to be T-cell mediated, Rock and colleagues have treated PSS patients with photopheresis.sup.4. In this trial, 56 patients were enrolled into a randomized non-blinded clinical trial. A significantly higher response rate was seen in the photopheresis treated group (68% response rate) compared to the D-penicillamine (control) group (32% response rate).
Juvenile onset diabetes mellitus (JODM) is felt to be mediated by the immune system resulting in the destruction of the cells in the pancreas responsible for the production of insulin. Patients with this disorder have not only dysregulation of their blood sugar levels, but the disease is characterized by a vasculopathy, resulting in specific organ damage leading to significant morbidity and mortality.
IBD is either limited to the colon (ulcerative colitis) or affects both the colon and the small intestine. In addition, there are intraintestinal manifestations of the disease including pyoderma gangrenosum, erythema nodosum, sclerosing cholangitis, ankylosing spondylitis, hepatitis, arthritis, uveitis. IBD involves a dysfunction of the immunoregulatory mechanisms that downregulate immune responses to digestion products, while maintaining the ability to develop a specific immune response to pathogens. Exposure to methoxsalen, activated by UV light, modulates immunoregulatory function, allowing the mucosal T-cenls to mount a lower proliferative response to common microbial antigens than periferal T-cells.
Other T-cell mediated phenomena include rejection of tissues that are foreign to the host. In the case of organ allograft transplantation, it is desirable to prevent this rejection with respect to the transplanted organ, however to otherwise maintain the competence of the immune system, in order to allow the body to combat infection and to allow other normal body defenses. The standard treatments after transplantation are limited as the immunosuppression regimens used to cause a state of general immunosuppression, which leads to the most common adverse reaction to this treatment, again infection, which may be microbial or opportunistic infection. Immunomodulation which does not have broad immunosuppressive properties would be more desirable. Photopheresis has been shown to be effective, and investigators at Loyola University have been able to successfully treat with photopheresis 13 of 14 cases of cardiac rejection refractory to standard immnunosuppressive agents. In a variation of this situation, photopheresis has been successfully used to treat a patient with chronic graft versus host disease.sup.5. This disorder is characterized by an introgenically induced immunoincompetent host, where immune competent cells (bone marrow or peripheral stem cells) are infused into a patient in such situations as treatment for various malignancies and leukemia. Here the transplanted immunocompetent cells attack the patient (the "host"), and the issue is to modulate the immunocompetent cells without causing further broad immunosuppression and the side effects thereof.
Photopheresis involves the extracorporeal exposure of peripheral blood leukocytes to 8-methoxypsoralen (8-MOP) photoactivated by ultraviolet A light, followed by the reinfusion of the treated white blood cells.
8-methoxypsoralen molecules in the blood enter the white blood cell nuclei and intercalate in the double-strand DNA helix. In an extracorporeal circuit, long wave ultraviolet light is directed at the leukocyte-enriched blood fraction within the UVAR.RTM. Photopheresis System. The photoactivated drug, responding to the UVA energy, links to the thymidine base in the DNA helix. This results in cross-linking of thymidine bases which prevents the unwinding of the DNA during transcription. The plasma and altered leukocytes are then reinfused into the patient. The reinfusion of the photopheresis damaged leukocytes results in an delayed immune attack against these damaged leukocytes, as well as, otherwise unmodified WBC's displaying the same cell surface antigens.
Methoxsalen is a naturally occurring photoactive substance found in the seed of the Ammi majus (umbelliferae plant). It belongs to a class of compounds known as psoralens or furocoumarins. The chemical name is 9-methoxy-7H-furo[3,2-g][1]-benzopyran-7-one. The formulation of the drug is a sterile liquid at a concentration of 20 mcg/mL in a 10 mL vial. The pharmacokinetic activity of methoxsalen is available in the investigator's brochure.sup.6. Toxicology studies of extracorporeal photochemotherapy and different dosages of UVADEX.RTM. and ultraviolet light in beagle dogs is lo located in the investigator's brochure.
UVAR System
The treatment consists of three phases including: 1) the collection of a buffy-coat fraction (leukocyte-enriched), 2) irradiation of the collected buffy coat fraction, and 3) reinfusion of the treated white blood cells. The collection phase has six cycles of blood withdrawal, centrifugation, and reinfusion steps. During each cycle, whole blood is centrifuged and separated in a pediatric pheresis bowl. From this separation, plasma (volume in each cycle is determined by the UVAR.RTM. Instrument operator) and 40 ml buffy coat are saved in each collection cycle. The red cells and all additional plasma are reinfused to the patient before beginning the next collection cycle. Finally, a total of 240 ml of buffy coat and 300 ml of plasma are separated and saved for UVA irradiation.
The irradiation of the leukocyte-enriched blood within the irradiation circuit begins during the buffy coat collection of the first collection cycle. The collected plasma and buffy coat are mixed with 200 ml of heparinized normal saline and 200 mg of UVADEX.RTM. (water soluble 8-methoxypsoralin). This mixture flows in a 1.4 mm thick layer through the PHOTOCEPTOR.RTM. Photoactivation Chamber, which is inserted between two banks of UVA lamps of the PHOTOSETTE.RTM.. PHOTOSETTE.RTM. UVA lamps irradiate both sides of this UVA-transparent PHOTOCEPTOR.RTM. chamber, permitting a 180-minute exposure to ultraviolet A light, yielding an average exposure per lymphocyte of 1-2 J/cm.sup.2. The final buffy coat preparation contains an estimated 20% to 25% of the total peripheral blood mononuclear cell component and has a hematocrit from 2.5% to 7%. Following the photoactivation period, the volume is reinfused to the patient over a 30 to 45 minute period.
Systems employing these techniques are known whereby extracorporeal treatment of a patient's blood is undertaken. For example, in U.S. Pat. No. 4,573,960--Goss, a patient is given a drug that requires photoactivation and the patient's blood is then withdrawn and separated into its components. The untreated components (red blood cells, some plasma, etc.) are returned to the patient. The patient is then disconnected from the treatment apparatus and the separated components, e.g., white blood cells, are exposed to ultraviolet light. Following photoactivation, the treated cells are returned to the patient.
In U.S. Pat. Nos. 4,321,919; 4,398,906; and 4,464,166, issued to Edelson, the external treatment methods for diseases in which there is a pathological increase of lymphocytes, such as cutaneous T-cell lymphoma, have been discussed. In these methods the patient's blood in the presence of a chemical or an antibody is irradiated with ultraviolet light. Ultraviolet light effects a bonding between the lymphocytes and the chemical or antibody thus inhibiting the metabolic processes of the lymphocytes.
A variety of human viruses are able to infect and replicate within mononuclear cells, or infectious viral particles may remain present within the mononuclear cells. The mononuclear cells can act as either a source for viral replication and spread of the virus, or as a reservoir of infectious virus particles which is difficult for the immune system to eliminate. Failure to eliminate these sources of infectious virus may lead to the establishment of a chronic condition. Viruses which can infect, replicate within, or reside in mononuclear cells include, but are not limited to, arthropod borne viruses, enteroviruses, paramyxoviruses (RSV), herpes viruses, cytomegalo-virus (CMV), Epstein-Barr virus (EBV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis G virus (HGV), and retroviruses (such as HIV).
A variety of human non-viral pathogenic agents are able to infect and replicate within mononuclear cells, or the infectious non-viral pathogenic agents may remain present within the mononuclear cells. The mononuclear cells can act as either a source for replication and spread of the non-viral pathogenic agents, or as a reservoir of infectious non-viral pathogenic agents which is difficult for the immune system to eliminate. Failure to eliminate these sources of infectious non-viral pathogenic agents may lead to the establishment of a chronic condition. Non-viral pathogenic agents which can infect, replicate within, or reside in mononuclear cells include, but are not limited to, bacteria such as arthropod-borne bacteria, mycoplasma species, and mycobacteria species, and parasites such as plasmodium species and other arthropod-borne parasites.
Extracorporeal photopheresis (ECP) has been successfully used to treat HIV infection (U.S. Pat. No. 4,960,408) and psoralen compounds with long wavelength ultraviolet light have been shown to inactivate certain viruses in vitro, such as HIV (Quinnan, G. V. et al., 1986, Transfusion, 26, pp 481; Bisaccia, A. et al., 1990, Am. Intern. Med., 113, pp 270; Bisaccia, A. et al., 1991, Ann. NY Acad. Sci., 636, pp 321), and influenza virus and herpes simplex virus (Redfield, D. C. et al., 1981; Infect. and Immun., 32, pp 1216). Bisaccia from Columbia University has studied ECP in a pilot trial as therapy for patients with AIDS-related complex. The rationale was that a combination of psoralen with UVA activation could damage HIV in vitro and that reinfusion of the damaged virus may initiate an immune response. The authors found that ECP produced an increase in the HIV-Ab production, increase in the CD8(+) lymphocytes, a decrease in the p24 antigen titer and the inability to culture HIV in 3 patients. Eleven of the 20 patients had improvement in their skin test antigen reactivities.
In addition, a reduced incidence of infection episodes was reported in patients receiving photopheresis treatment for immunosuppression following transplant surgery (Meiser, B. M. et al., 1994, Transplantation, 57, pp. 563). However, the results observed for the transplant surgery patients did not correlate with photopheresis treatment since infection episodes in general were recorded including patients who received a variety of treatments to prevent rejection of the transplanted organ.
Hepatitis C is a common and major cause of serious liver disease and cirrhosis. Approximately 200 million people in the world are currently estimated to be infected with the hepatitis C virus. The prevalence in the United States is estimated to be 0.6 to 1.0% of the population or approximately 3.5 million people.sup.7. It is reported that chronic hepatitis develops in at least half of those patients with acute hepatitis C. 20% of those will develop liver cirrhosis.sup.8,9.
The HCV genome is a small, enveloped, single stranded, positive-sense RNA virus.sup.10. It resembles viruses in the Flaviviridae family, both the flaviviruses (dengue and yellow fever viruses) and the pestiviruses.sup.11. The HCV genome contains one large open reading frame encoding approximately 3000 amino acids.sup.12.
The 5' end starts with a non-coding region. The next structures are the C region which encodes the core of the virus, E1 and E2 are the envelope glycoproteins of the virus. The E2/NS 1 region contain a hypervariable region. Mutations within this area appear to change outer envelope epitopes resulting in escape from immune recognition. Regions NS2 through NS5 all encode nonstructural proteins necessary for viral replication.
The RNA-dependent RNA polymerase has no proof-reading ability. This results in a high error rate during replication causing genomic variants to form. A consensus system for genotype classification of the hepatitis C virus has recently been developed. This classification system acknowledges at least six major genotypes of hepatitis C (genotypes 1-6) present worldwide.sup.13. Each major genotype is further subdivided into 0-3 minor genotypes designated a, b and c. Genotypes 1a (35%) and 1b (35%) are the most common genotypes in the United States. In Western Europe, types 1, 2 and 3 are common. Type 4 is mainly found in Africa and type 5 is associated with Dutch ancestry and is present in South A-frica and the Netherlands. Type 6 is prevalent in Hong Kong. Infection by HCV variants have different rates of progression to cirrhosis, sensitivity to interferon and possibly, to rate of development to hepatocellular carcinoma.
The application of the PCR technique to amplify reverse-transcribed DNA provides a very sensitive assay for detection viral RNA. PCR assays have been able to detect HCV RNA within only a few days of exposure to the virus, weeks before elevations of viral antibody levels.sup.14,15. This assay can be used to directly monitor the antiviral effect of therapy.sup.16.
The fundamental defect that allows establishment of chronic hepatitis C following acute HCV infection is not known. Several investigators have suggested that impaired cellular immunity, either T cells or natural killer cells, may play a role. Weiner et al. have demonstrated the existence of a hypervariable region of the HCV genome in the E2/NS 1 segment.sup.17. This area codes for isolate-specific, B-cell antibody binding linear epitopes that are expressed on the envelope surface of the HCV particle. The characteristics of this domain are similar to the V3 loop of HIV-1's gp 120 protein. The rapid mutation within this region may explain a loss of immune recognition and clearance of the hepatitis C virus.
The basic immune mechanism which results in the clearance of hepatitis C virus is still not known. Shirai from the National Cancer Institute has demonstrated that CD8(+) cytotoxic lymphocytes recognize a nonstructural protein with homology to RNA polymerase expressed in association with an HLA class I antigen on the surface of the hepatocytes.sup.18. The hepatitis C virus is a positive strand virus that replicates by producing a negative-strand RNA as a template. During active HCV replication, these negative-strand RNA templates are present in the patient's liver. Investigators have also found the presence of active, replicating hepatitis C viral particles in the patient's peripheral blood mononuclear cells.sup.19. Monocytes, macrophages, T-cells and B-cells can all be shown to contain negative-strand HCV RNA.
Subtle changes in monocyte function have been observed in patients chronically infected with HCV as compared to matched controls. In this study by Mendoza and others, .sup.20 PBMC and purified monocytes from HCV-seropositive patients and uninfected controls were stimulated with PMA. Supernatants from the chronic HCV carriers secreted less IL1 and TNF compared to matched uninfected controls. These findings suggest that functional impairment of macrophages may exist during chronic HCV infection and may be the mechanism by which HCV infection persists.
During therapy with alpha interferon, hepatitis C virus can disappear from the patient's liver and blood as measured by RT-PCR. Despite this apparent clearance of virus, there is a very high (80-20 85%) relapse rate after interferon therapy. Investigators have demonstrated the (few) patients who do not have HCV replication detected in the PBMCs at the end of therapy, do not relapse after interferon therapy and are apparent cures.sup.21,22,23.
It would appear that the mononuclear cell can serve as an immunologically protected site that shelters the hepatitis C virus from immune system recognition and attack. Once therapy with alpha interferon is withdrawn, the virus may leave the PBMCs and reinfect the patient's blood and liver.